The present invention pertains to the fields of plant molecular biology. More specifically, the invention relates to induction of systemic acquired disease resistance in plants involving a novel salicylic acid-independent signal transduction pathway whose activation is associated with enhancement of a plant""s ability to resist microbial infection.
Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
In plants, resistance to environmental challenges, e.g. pathogens, insects and stresses, enables them to better survive in nature, Resistance to pathogen attack is often associated with the hypersensitive response (HR), in which rapid, local cell death occurs at the infection site. The formation of these necrotic lesions is associated with the restriction of pathogen multiplication and spread. In the tobacco/tobacco mosaic virus (TMV) system, a typical HR develops on TMV-inoculated leaves of the tobacco cultivar Xanthi nc, which contains the N resistance gene. By contrast, when the TMV-susceptible cultivar Xanthi is infected, the virus spreads throughout the plant and causes disease symptoms. Several days after HR formation, systemic acquired resistance (SAR) develops throughout the plant (Ross, 1961a, 1961b; Ryals et al., 1994, 1996). SAR confers enhanced resistance not only to a secondary challenge by the initial infecting pathogen, but also to a broad range of other pathogens. Therefore, plant disease resistance is associated with both local (HR) and systemic (SAR) responses.
Activation of resistance responses is associated with the induction of a large number of defense genes and the synthesis of many secondary compounds. The former include genes encoding peroxidases, glucanases, chitinases, and other pathogenesis-related (PR) proteins while the latter include phytoalexins, phenolic compounds and lignin (Bowles, 1990; Cutt and Klessig, 1992). Phytoalexins are low molecular weight antimicrobial compounds that are synthesized by the plant and accumulate at the site of infection. While most phytoalexins are synthesized via the phenylpropanoid pathway, some are synthesized by the isoprenoid biosynthetic pathway (Ebel, 1986). Phenylalanine ammonia-lyase (PAL) is the first enzyme in the phenylpropanoid pathway and it regulates the biosynthesis of flavonoids, phytoalexins and lignins (Hahlbrock and Scheel, 1989; Dixon and Lamb, 1990). Levels of PAL mRNA and enzymatic activity increase in the inoculated tissue of resistant plants after pathogen infection. In addition, when tobacco plants are treated with a fungal elicitor, PAL mRNA accumulates in stem tissues (Pellegrini et al., 1994).
Plants resisting pathogen attack also synthesize a variety of PR proteins (Linthorst, 1991; Cutt and Klessig, 1992). In tobacco, at least five families of PR genes have been identified. They are induced in both the TMV-infected leaves and the upper uninoculated leaves of resistant cultivars (Ward et al., 1991). The function of some PR proteins is still unclear; however, several have been shown to have antimicrobial activities either in vivo or in vitro (Dempsey and Klessig, 1995).
Increases in salicylic acid (SA) levels also correlate with resistance to pathogen attack (Malamy et al., 1990; Metraux et al., 1990; Rasmussen et al., 1991). In tobacco resisting TMV infection, endogenous SA levels increase in both inoculated and uninoculated leaves. These increases correlate with the HR and SAR and precede the induction of PR genes (Malamy et al., 1990). A correlation between SA accumulation and resistance to pathogen attack has also been shown in other plants, e.g. cucumber and Arabidopsis (Metraux et al., 1990; Rasmussen et al., 1991; Uknes et al., 1993; Lawton et al., 1994; Summermatter et al., 1995; Dempsey et al., 1997). In addition, exogenously applied SA enhances resistance and induces nine gene families whose expression is associated with SAR in TMV-inoculated tobacco (Ward et al., 1991). Elevated SA levels also correlate with the activation of plant defenses in temperature shifted tobacco. At temperatures above 28xc2x0 C., development of resistance to TMV is blocked and tobacco plants become systemically infected (Kassanis, 1952). At this elevated temperature, there is no detectable increase in SA levels, no PR protein synthesis and no HER after TMV infection (Gianinazzi, 1970; Yalpani et al., 1991; Malamy et al., 1992). However, when these infected tobacco plants are shifted to lower temperatures, resistance to TMV is restored. Moreover, a rapid and dramatic increase in SA levels precedes both PR gene expression and the appearance of a HR (Malamy et al., 1992).
Mutant analysis in Arabidopsis also provides support for SA""s importance in disease resistance. Several mutants (e.g. npr1, nim1, and sai1), have been isolated which are unable to activate PR gene expression after treatment with SA or INA (2,6-dichloroisonicotinic acid, a synthetic functional analog of SA; Conrath et al., 1995; Durner and Klessig, 1995; Vernooij et al., 1995; Malamy et al., 1996). These mutants show greater susceptibility to bacterial and fungal pathogens (Cao et al., 1994; Delaney et al., 1995; Shah et al., 1997). conversely, mutants with elevated levels of SA, including the lesion mimic mutants acd2 and the 1sd series (Dietrich et al., 1994; Greenberg et al., 1994), as well as cpr1 (Bowling et al., 1994) and cep1 (Klessig et al., 1996), constitutively express these PR genes and exhibit enhanced resistance to pathogens.
Some of the strongest evidence supporting a signaling role for SA comes from the analysis of transgenic tobacco and Arabidopsis plants that contain the bacterial nahG gene encoding salicylate hydroxylase. Since salicylate hydroxylase degrades SA, these transgenic NahG plants are unable to accumulate SA. Following inoculation with TMV, NahG tobacco plants exhibit substantially reduced induction of PR gene expression and fail to develop SAR. Furthermore, they show enhanced susceptibility to infection with both virulent and avirulent pathogens (Gaffney et al., 1993; Delaney et al., 1994; Vernooij et al., 1994). Thus, SA appears to be required for an effective resistance response.
While SA mediates resistance to certain pathogens, other signal transduction pathways also appear to be involved in plant defense response. Both ethylene and jasmonates have been implicated as signal molecules that induce PR proteins during plant defense responses (Boller et al., 1983; Boller, 1991; Xu et al., 1994). Furthermore, several recent reports have shown that induction of a number of defense genes by pathogens, elicitors or abiotic agents (soluble sugars) is mediated by a SA-independent pathway(s) (Herbers et al., 1996; Penninckx et al., 1996; Pieterse et al., 1996; Vidal et al., 1997).
Compositions and methods that enhance disease resistance in plants are agronomically important and highly desirable. While SA is a key component in a plant""s capacity to withstand environmental stresses, mounting evidence suggests that a second pathway leading to systemic acquired disease resistance exists. Thus, a need exists to identify and characterize the genes and encoded proteins involved in this novel, second pathway.
The present invention relates to a new signal transduction pathway in plants which is associated with and leads to the development of systemic acquired resistance (SAR). Activation of this pathway results in the induction of a diverse group of genes which are expressed in both uninfected and infected tissues in a salicylic acid (SA)-independent manner. These genes are termed SIS for SA-independent systemically induced.
According to one aspect of the invention, a method for identifying SIS genes is provided. The method uses the following steps: (a) providing pairs of plants, each pair being of of equivalent species and variety, wherein one of the pair (the xe2x80x9cSA+xe2x80x9d plant) accumulates endogenous SA, and the other of the pair (the xe2x80x9cSA-xe2x80x9d plant) does not accumulate endogenous SA; (b) inoculating one pair of plants with a plant pathogen to which the plant variety responds by exhibiting SAR; (c) mock-inoculating another pair of plants; (d) identifying defense-related genes in the plants by detecting genes that are expressed in the inoculated plants but not in the mock-inoculated plants (the defense-related genes indentified in this manner include the SIS genes as well as other defense-related genes); and (e) identifying the SIS genes by detecting the defense-related genes expressed in the inoculated SA+ plant and the defense-related genes expressed in the inoculated SAxe2x88x92 plant, the SIS genes being expressed in both the inoculated SA and SA plants, the other defense-related genes being expressed only in the inoculated SA+ plant. In a preferred embodiment, the pair of plants are tobacco of the variety Xanthi nc and the pathogen is TMV. The plant not accumulating SA expresses a nahG gene, resulting in conversion of SA to catechol.
According to another aspect of the invention, a method is provided for identifying compounds that activate an SI-SAR pathway in plants. The method has the following steps: (a) providing a first DNA construct in which a first reporter gene is operably linked to a SIS gene promoter; (b) providing a second DNA construct in which a second reporter gene is operably linked to a SA-inducible gene promoter, such as a PR-1 gene promoter; (c) transforming a plant with the first and second DNA constructs; (d) administering to the plant a test compound suspected of activating the SI-SAR pathway; and (e) detecting expression of the first and second reporter genes, expression of the first reporter gene and lack of expression of the second reporter gene being indicative of activation of the SI-SAR pathway by the test compound.
Another method for identifying compounds that activate an SI-SAR pathway in plants comprises the following steps: (a) providing a plant that does not accumulate endogenous SA; (b) transforming the plant with a DNA construct in which a reporter gene is operably linked to a SIS gene promoter; (c) administering to the plant a test compound suspected of activating the SI-SAR pathway; and (d) detecting expression of the reporter gene, the expression of the reporter gene being indicative of activation of the SI-SAR pathway by the test compound.
According to another aspect of the invention, a method is provided for enhancing SA-independent resistance of a plant to infection by a plant pathogen. The method comprises transforming the plant with a DNA construct having a coding region of at least one SIS gene operably linked to a constitutive promoter, causing the transformed plant to constitutively express the SIS gene, thereby conferring the enhanced resistance to the plant pathogen.
In another aspect of the invention, a method for isolating signaling components of the SI-SAR pathway in plants is provided. The method comprises: (a) transforming a plant with a DNA construct having a reporter gene coding sequence operably linked to a SIS gene promoter; (b) producing progeny of the transgenic plant that are homozygous for the transgene; (c) mutagenizing seeds of the progeny; (d) producing M2 progeny from the mutagenized seeds; (e) selecting individuals of the M2 progeny that desplay altered expression of the reporter transgene as compared with non-mutagenized plants; and (f) identifying mutations in the selected mutagenized plants that confer the altered expression of the reporter transgene, the mutations being associated with genes that: encode signaling components of the SA-SAR pathway.
According to another aspect of the present invention, an isolated nucleic acid molecule is provided that includes an open reading frame encoding a plant pathogen-inducible epoxide hydrolase (piEH). This nucleic acid represents one of the SIS genes identified in accordance with the invention. In a preferred embodiment, the nucleic acid encodes a piEH that is inducible in the absence of SA. In various embodiments of the invention, the nucleic acid is DNA, either cDNA or genomic DNA. In other embodiments, it is RNA. In a preferred embodiment, the nucleic acid encodes a tobacco piEH, preferably having an amino acid sequence which is greater than 40% homologous with Sequence I.D. No. 2, and most preferably comprises Sequence I.D. No. 2.
According to another aspect of the invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (a) Sequence I.D. No. 1; (b) an allelic variant of Sequence I.D. No. 1; (c) a natural mutant of Sequence I.D. No. 1; (d) a sequence hybridizing with part or all of a sequence complementary to Sequence I.D. No. 1 and encoding a polypeptide substantially the same as part or all of a polypeptide encoded by sequence I.D. No. 1; and (e) a sequence encoding part or all of a polypeptide having amino acid sequence I.D. No. 2.
Also provided in accordance with the invention are recombinant DNA molecules comprising the aforementioned nucleic acid molecules, operably linked to vectors for transforming plant cells. Also provided are plant cells transformed with those recombinant DNA molecules, and transgenic plants comprising those recombinant DNA molecules.
According to another aspect of the invention, an isolated plant pathogen-inducible epoxide hydrolase (piEH) is provided, the expression of which is inducible by inoculation of the plant with a pathogen, in the absence of salicylic acid. In a preferred embodiment, the piEH is of tobacco origin and is inducible by inoculation of tobacco with TMV. In preferred embodiments, the piEH has an amino acid sequence greater than 40% homologous to Sequence I.D. No. 2, and most preferably comprises Sequence I.D. No. 2.
According to another aspect of the invention, a polypeptide is provided, which is produced by expression of an isolated nucleic acid sequence selected from the group consisting of: (a) Sequence I.D. No. 1; (b) an allelic variant of Sequence I.D. No. 1; (c) a natural mutant of Sequence I.D. No. 1; (d) a sequence hybridizing with part or all of a sequence complementary to Sequence I.D. No. 1 and encoding a polypeptide substantially the same as part or all of a polypeptide encoded by sequence I.D. No. 1; and (e) a sequence encoding part or all of a polypeptide having amino acid sequence I.D. No. 2.
In another aspect of the invention, antibodies immunologically specific for part or all of the aforementioned proteins are provided.
Other aspects and advantages of the invention will be understood by reference to the detailed description of the invention and examples set forth below.